Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
Applied Veterinary Bacteriology and Mycology:
Bacteriological techniques
Chapter 7: Bacterial cell counting techniques
Author: Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION ......................................................................................................................................................2
VIABLE COUNTING METHODS .............................................................................................................................2
1|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
INTRODUCTION
Bacterial cell counts are used in diagnostic and food hygiene laboratories. In a diagnostic laboratory, they are
used to enumerate bacterial cells in fluids such as autogenous vaccines, semen, water, milk or urine.
Both viable and total counts can be carried out. Viable counting techniques are more commonly used. Viable
bacteria are capable of multiplication with the production of visible colonies on or in agar media. In viable
counting methods, the assumption is made that one well-spaced, bacterial cell gives rise to one colony. Total
counts will enumerate both viable and non-viable bacterial cells.
VIABLE COUNTING METHODS
For all viable bacteria counting methods, serial ten-fold dilutions are made of the original sample, which contains
the bacteria to be counted. The diluent is either saline or an appropriate nutrient broth.
1.
Spread plate method
A range of ten-fold dilutions is used and from each dilution an inoculum of 0,1 ml is pipetted onto the surface of
separate agar plates. The inoculum is spread rapidly over the entire agar surface using a thin bent glass rod
(bent Pasteur pipette) or flame-sterilized nichrome wire bent in an L-shape. The glass rod can be sterilized by
placing it in 96% ethanol or methanol and then flaming it until all the alcohol has evaporated. At least two, but
preferably four plates should be inoculated per dilution. For viable counts of Escherichia coli, plate count agar,
nutrient agar or MacConkey agar can be used. The plates are incubated for 24-48 hours at 25 - 37°C dependent
on whether environmental (25°C) or pathogenic (37°C) bacteria are sought. After incubation, plates inoculated
with a sample dilution yielding between 30 and 300 colonies are read. The colony count should be an average of
two or four plates inoculated with the selected dilution. Various instruments, such as electronic counters, are
available to facilitate the counting of colonies.
2.
Pour plate method
This method is similar to the spread plate technique, except that 0,1 ml of each dilution is mixed thoroughly in
molten agar, held before use in a water bath at 50°C and poured into separated 90mm Petri dishes. This
procedure should be repeated for each dilution 2 to 4 times. The agar is allowed to set and then incubated at 25
or 37°C for 24 - 48 hours. Plates inoculated with a sample dilution that yields between 30 to 300 colonies per
plate should be read. The colonies will be well distributed throughout the agar as well as on the surface. The
subsurface colonies of most genera of bacteria assume a biconvex shape.
3.
Calculation for the number of colony forming units (CFU) when using the spread and pour plate
methods
Number of CFU/ml = N x 10n x 10
2|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
N = no. of colonies on the plate at the selected dilution n.
For example, if there is a mean count of 250 colonies per plate at the 10 -4 dilution and the inoculum was 0,1 ml
per plate:
The number of CFU/0,1 ml of original sample = 250 x 104.
Thus, the number of bacteria/1ml of original sample = 250 x 10 4 x 10 = 2,5 x 107.
Preparation of ten-fold dilutions for a viable bacterial count. The specimen and each dilution must be thoroughly
mixed before sampling and a separate pipette must be used for each transfer step, to prevent carry-over of
bacteria.
Figure 1: 10-fold serial dilution method used for bacterial counts
4.
Miles-Misra technique
This method has the advantage of being economical with agar media. Lines are drawn on the bottom of an agar
plate, dividing it into 8 sectors. An inoculum of 0,02 ml from each ten-fold dilution is pipetted as a drop on the
agar in each sector. At least 4 separate drops per sample dilution should be used. The inocula are allowed to dry
and the plates are incubated at 25 or 37°C for 24 - 48 hours. A sample dilution yielding about 30 colonies per
drop should be selected. An average count from at least 4 drops should be obtained. The calculation is similar to
the previous two methods, but as the inoculum was 0,02 ml, the conversion factor will be 50 instead of 10, to
obtain a figure for the CFU/ml in the original sample.
5.
Filtration method
This is a useful method for determining the number of bacteria in a water sample or other clear fluid in which the
bacterial number is low. A known volume of water is passed through a membrane filter of pore size 0,22 µm. The
filter will retain the bacterial cells, and is aseptically placed, bacterial-side up on the surface of an agar plate. The
medium can be selective or non-selective depending on the bacterial species being sought. Colonies will form
on the surface of the filter after 24-48 hours of incubation and can be counted. As the volume of fluid is known,
the bacteria/ml or per 100 ml of sample can be calculated.
3|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
6.
Surface contact plates
Special plastic plates (RODAC plates) containing various agar media are available to allow direct sampling of flat
surfaces for the presence of bacteria. The technique can be used to detect a specific pathogen, such as
Salmonella when a selective medium is used, or to determine the degree of contamination of a surface using a
non-selective medium. An exact quantity of agar medium must be used to fill these plates as the agar surface
should project slightly above the rim of the plate. Surfaces are sampled by placing the agar gently on the area,
the plate lifted carefully and the lid replaced. Another method is by the use of an agar sausage medium, which is
cut into discs. After touching the test surface with the base of the disc, the latter is placed in a 60 mm diameter
plastic Petri dish, which is marked on its lower surface with a grid. Plates are incubated at 25 or 37°C for 24 - 48
hours. The number of bacteria/cm 2 can then be calculated, using the grid at the base of the plate.
7.
Quantitative tissue cultures
Quantitative tissue cultures are rarely requested in veterinary medicine. They are used for the evaluation of the
clinical significance of a bacterial isolate(s) recovered from tissue. The presence of >10 5 colonies/gram of tissue
is thought to be tissue colonisation rather than tissue contamination by bacteria. The following protocol may be
used for performing quantitative bacterial cultures:
1. Weigh the sterile, empty specimen container.
2. Weigh the tissue in the container.
3. Mince the tissue with sterile scissors, and then grind it with a pestle in a mortar containing 1 ml of nutrient
broth per g of tissue or in a tissue homogenizer containing a similar volume of nutrient broth.
4. Dilute the suspension as follows:
4.1 0,9 ml nutrient broth + 0,1 ml of suspension (=10-1)
4.2 0,9 ml nutrient broth + 0,1 ml of 10-1 suspension (=10-2)
4.3 0,9 ml nutrient broth + 0,1 ml of 10-2 suspension (=10-3)
5. Inoculate 0,1 ml of each dilution onto a blood agar plate. Spread the inoculum evenly over the surface of the
agar with a sterile bent glass rod (“hockey stick”).
6. Quantitate the growth as follows:
organisms/gram of tissue = N x D x F x10
W
N = number of colonies on the agar plate selected for enumeration
D = reciprocal of the dilution inoculated onto the agar.
4|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
W = Weight of tissue in grams
F = dilution factor.
V= Volume of the broth used to suspend the tissue
8.
Total aerobic bacterial count using the Petrifilm TM method
Introduction
The total aerobic bacterial count method tests for all saprophytic bacteria (Gram-positive and Gram-negative)
present in water or milk samples.
The 3M PetrifilmTM Aerobic Count is used in this test and is a ready-made culture medium system that contains
nutrients, a cold-water-soluble gelling agent, and tetrazolium indicator that facilitates colony enumeration.
Petrifilm AC is useful for the enumeration of aerobic bacteria in water samples and is decontaminated though not
sterilized.
Materials/ sample:

Petrifilm Aerobic Count (Code: 6400/6406) Merck NT Laboratory Supplies

1 ml disposable pipette (Code: 40101) Adcock Ingram Critical Care BP or pipette tips

Pipette Boy, pipette suction bulb or micropipette (1000 µl)

Plastic spreader

Desk top incubator
Method:

Place the Petrifilm on a level surface.

Lift the top film and dispense 1 ml of the water sample to the center of bottom film.

Drop the top film onto sample.

With the recessed side down, place the plastic spreader on the center of the plate.

Distribute the sample evenly by pushing gently downward on the center of the plastic spreader.

Do not slide the spreader across the plastic film.

Remove the spreader and leave the plate undisturbed for at least one minute to permit the gel to solidify.

Incubate plate in a horizontal position with the clear side up for 48 hours. Plates can be incubated in stacks
up to 20.
Interpretation:

Count all red colonies regardless of size or intensity.

The circular growth area is approximately 20 cm 2. Estimates can be made on plates containing greater
than 250 colonies by counting the number of colonies in four squares and multiplying the total by 5 to
determine the total count per film.

The count should be interpreted as TNTC (too numerous to count) if the following occurs:
5|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods

9.

High concentrations of colonies on the plate will cause the entire growth area to become red or pink;

The center may lack visible colonies but many small colonies can be seen on the edges;

The colonies are too many and close together to count
The final count should be noted as total count/ ml water.
Use of marker bacteria
Occasionally investigations of infectious agents may require the use of a marker organism. Serratia rubidea is
most commonly used, as it is not considered to be a pathogen and its colonies are a bright red. The marker
bacteria employed should have similar characteristics to the organisms being investigated. This method can be
used for testing the efficacy of a disinfection programme, or studying the dispersal of pathogenic microorganisms as an aerosol, or in the determination of the efficacy of sewage treatment.
10. Most probable number (MPN) techniques
These techniques are based upon statistical probabilities with the assumption that there is a uniform distribution
of bacteria in liquid or homogenized samples. If a liquid sample contains one viable bacterial cell, its growth and
multiplication in a suitable broth can be detected by manifestations such as turbidity or acid and gas production.
These methods can be used for most bacteria, but they are commonly used for the detection of coliform bacteria
in water supplies or for the quantification of antimicrobials in a specified sample. MacConkey broth with
bromocresol purple as pH indicator is often used in coliform counts. Acid production is indicated by a yellow
colouration of the broth and gas is trapped in a Duram tube.
Procedure:
1.
Prepare at least 5 ten-fold dilutions of the sample, with three, five or ten duplicates of each dilution. The
samples should be placed in an appropriate nutrient broth in a 1:10 ration. For water analysis, a double
strength broth is used with an equal volume of the sample to avoid excessive nutrient and inhibitor dilution.
2.
3.
Incubate the tubes at 35°C for 48 hours and examine each for acid and gas production.
By referring to standard MPN probability tables, the MPN of coliforms/100 ml of water can be determined.
10.1 Total coliform and E. coli counts using CollilertTM
Introduction
The presence of coliform bacteria in water is regarded as a warning signal that the water is subject to potentially
dangerous pollution. The coliform group of bacteria includes all the aerobic, facultative anaerobic, gram negative
and non-sporulating bacilli that produce acid and gas from the fermentation of glucose. The classic species of
this group are Escherichia coli and Enterobacter aerogenes. The other organisms of this group are: Salmonella
spp, Shigella spp., Proteus spp, Pseudomonas spp. and Alcaligenes spp.
6|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
The Collilert product is used in this test. Collilert – 18 simultaneously detects total coliforms and E. coli in water.
When total coliforms metabolize Collilert 18’s nutrient – indicator, ONPG, the sample turns yellow. When E. coli
metabolize Collilert – 18’s nutrient – indicator, MUG, the sample fluoresces.
Materials/ sample:

100 ml measuring cylinder

100 ml Schott bottle

Collilert Pres ABS (Code: 04 – 021373) (Dehteq)

10 x 10 ml sterile glass tubes

10 ml disposable pipette (Code: 47110) Adcock Ingram Critical Care BP

Incubator at 37 °C

5 ml plastic tubes Code: AS 036 Plaspro Scientific (PTY) LTD

UV light
Method:

Measure 100 ml of sample and place in Schott bottle

Add contents of one CollilertTM ampoule to sample

Shake well until dissolved

Transfer mixture to 10 glass tubes each with 10 ml

Incubate all tubes for 18 hours at 37°C
Interpretation:
Coliforms: Count the number of positive/ yellow tubes and refer to the MPN table; the final count should be noted
as cfu/ 100 ml water
E. coli: Transfer all positive samples to 5 ml plastic tubes and place on the UV light, a purple fluorescense
indicates the presence of E. coli.
11. Bioluminescence ATP assay method for the rapid detection of bacteria
Use of firefly luciferase to assay adenosine triphosphate (ATP) extracted from microorganisms provides an easy
means to enumerate microbes within minutes. The method has also been used to more rapidly detect the growth
of slow-growing bacteria and fungi and antimicrobial susceptibility assays. The small amount of light produced is
proportional to ATP and thus microbial number. The average bacterium contains around 10 -15 g ATP per cell.
Present reagents permit detection of 103 cells per tube. Luminometers currently on the market detect about 10 -12
g ATP. Proper extraction of ATP from the microbes is an essential part of any protocol, as is the removal of nonmicrobial ATP from, for example, somatic cells also present in samples. The technique may be applied to a wide
range of samples, for example food and beverages and clinical samples such as urine. The ATP assay gives a
global measure of microbial numbers, i.e. it is not species specific unless a species separation step is included in
the protocol. This method is not always accurate for mixed bacterial populations as different bacteria have
7|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
different extraction criteria. It can also give viable results when both stationary bacteria as well as those in the
log phase of growth are present.
Total counts of bacterial cells
These counts will include living and dead cells.
1.
Turbidity standards
Brown’s or McFarland’s opacity tubes are available commercially or can be made using barium sulphate. They
consist of a series of ten numbered, standard, thin glass tubes containing different dilutions of suspended barium
chloride/sulphuric acid or barium sulphate, giving a range of opacities. The test bacterial suspension is placed in
an empty tube of similar dimension to the standards. A visual comparison of the opacities is made by rolling the
test suspension across a printed page and matching it with a standard of comparable opacity. Tables are
supplied with opacity tubes that give the numerical equivalents (bacteria/ml). It is a convenient and simple
method, but gives only an approximate total bacterial count. These methods are most commonly used for
bacterial suspensions to be used in the inoculation of biochemical tests and for antimicrobial sensitivity tests.
2.
Coulter counter
Coulter counters are automated, electronic counting instruments, usually used in haematology and in diary
science to enumerate mammalian cells, but they can be adjusted to conduct total bacterial cell counts.
3.
Breed’s direct smear method
This technique is most commonly used for counting bacteria in milk. A grease-free microscope slide is placed
over a template of a square of10 mm x 10mm, and 0,01 ml of sample is carefully spread over this area. The
smear is allowed to air-dry, fixed by heat and stained with methylene blue for 1 minute. After air drying the
stained smear is examined under the 100x oil-immersion objective of a microscope. The bacteria cells should be
counted in at least 50 fields throughout the area of the smear. The average bacterial cell count per field (N)
should be calculated. The radius (r) for the particular microscope’s oil immersion field can be determined (in mm)
using a slide and an eyepiece micrometer. This is usually about 0,08 mm, so that the area of the field, calculated
using the following formula, 3.14 x r2 (mm2), will be 0,25 mm2. Bacteria/ml in sample = N x 4 x 104.
N x area of smear (100 mm2 ) x 100
Area of one field (3,14 x r2)
Quantitative bacterial count of tissue on a Gram-stained smear:
1.
Transfer 0,1ml of undiluted tissue suspension from "quantitative tissue cultures, step 4.4" to a clean glass
microscope slide and spread it over a circular area not exceeding 15mm in diameter.
2.
Dry in an oven (75°C) for 15 minutes or air dry. Fix with concentrated methanol.
3.
Gram stain.
4.
Examine all fields under oil immersion (1000 X magnification).
8|Page
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Plating
Methods
Finding one or more bacteria in any field (not per oil immersion field) denotes the presence of at least 105
bacteria per gram of tissue.
4.
Counting chamber method
A Neubauer haemocytometer or Helber chamber can be used. To prevent motility of the bacteria, 2-3 drops of
full strength formalin/10 ml of bacterial suspension to be counted can be added.
The haemocytometer or chamber is filled and viewed under the low-power objective of a microscope in order to
orientate the marked grid. The 100X oil-immersion objective is used to count the bacteria in the five areas
marked with a square (Figure 24) in the central area of the grid. Each of these areas is divided into 16 smaller
squares. Thus the bacterial cells are counted in 80 (5 x 16) of the smaller squares. The average number of
bacteria (N) per small square can be calculated. The volume over each small square is:
Helber chamber: 0,0025 mm 2 x 0,1 mm depth = 0,00005 mm 3 .
Neubauer haemocytometer: 0,0025 mm 2 x 0,1mm depth = 0,00025 mm 3 .
Bacteria /ml in sample =
N x 1 x 1000 = N x 20 000 000 (Helber)
0,00005
N x 1 x 1000 = N x 4 000 000 (Neubauer)
0,00025
C
C
C
C
C
Figure 2: Grid of a Neubauer haemocytometer
9|Page